Method for the synthesis of evoc probes

Abstract

A process for the synthesis of a glycoside, comprising the step of;performing hydrolysis of a methyl ester glycoside in the presence of a base in water, to form a glycoside,wherein the propensity to reform the methyl ester glycoside is reduced. The invention also extends to methods for the detection or prognosis of cancer using a glycoside obtained by said process

1 . A process for the synthesis of a glycoside, comprising the step of;
 performing hydrolysis of a methyl ester glycoside in the presence of a base in water, to form a glycoside,   wherein the propensity to reform the methyl ester glycoside is reduced.   
     
     
         2 . The process according to  claim 1  wherein the base is NaOH, LiOH, KOH. 
     
     
         3 . The process according to  claim 1 or 2 , wherein the glycoside is selected from glucuronide, iduronide, mannoside, glucosamide, galactosamide, glucoside, galactoside, rhamnoside, riboside, arabinoside, fructoside, xyloside, fucoside. 
     
     
         4 . The process according to any one of  claims 1-3 , wherein the glycoside comprises a glycosidic bond linked to a volatile functional group. 
     
     
         5 . The process according to  claim 4 , wherein the glycosidic bond is selected from an O-, N-, S-, C-glycosidic bond. 
     
     
         6 . The process according to  claim 4 or 5 , wherein the volatile functional group is labelled. 
     
     
         7 . The process according to  claim 5 , wherein the label is selected from 12C, 13C, 14C, 2H, 14N or 18O. 
     
     
         8 . The process according to any one of  claims 4-7 , wherein the volatile functional group is selected from methyl, ethyl, propyl, isopropyl, butyl, methyl-D3, ethyl-D5, or propyl-D7. 
     
     
         9 . The process according to any one of  claims 1-8 , further comprising a step of ion-exchange after the step of hydrolysis, preferably wherein the ion-exchange is performed using an acidic cation exchange resin. 
     
     
         10 . The process according to any one of  claims 1-9 , further comprising a step of evaporating the solvent, after the step of hydrolysis. 
     
     
         11 . The process according to any one of  claims 1-10 , wherein the method comprises monitoring the hydrolysis of the methyl ester glycoside to form the glycoside, using  1 H NMR. 
     
     
         12 . The process according to any one of  claims 1-11 , wherein after the step of hydrolysis, the methyl ester glycoside is present at <1% of the glycoside. 
     
     
         13 . The process of claim any one of  claims 1-12 , wherein the hydrolysis is performed at a temperature between 18 to 30° C. 
     
     
         14 . The process according to any one of  claims 1-13  wherein the hydrolysis is performed under ambient conditions comprising a temperature of 25° C. (298.15 K) and pressure of 101.325 kPa. 
     
     
         15 . The process according to any one of  claims 1-14  wherein the methyl ester glycoside is D5-ethyl-βD-glucuronide-methyl ester, and wherein the glycoside is D5-ethyl-BD-glucuronide. 
     
     
         16 . A glycoside obtained by the process of any one of  claims 1-15 . 
     
     
         17 . A composition comprising a glycoside according to  claim 16 . 
     
     
         18 . A composition comprising a glycoside comprising a glycosidic bond linked to a volatile functional group, wherein the composition comprises <1 wt % of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group. 
     
     
         19 . A composition according to  claim 17 or 18 , wherein the <1 wt % of a methyl ester glycoside comprising a glycosidic bond linked to a volatile functional group, is determined via  1 H NMR. 
     
     
         20 . A glycoside according to  claim 16  for use in the diagnosis or prognosis of cancer comprising administering to a subject the glycoside according to  claim 16  or the composition according to any one of  claims 17-19 . 
     
     
         21 . A glycoside for use according to  claim 20 , wherein the glycoside is administered at a concentration of 0.05 to 10 mg/kg. 
     
     
         22 . A glycoside for use according to  claim 20 or 21 , wherein administration is intravenous and the concentration is 0.5 to 10 mg/kg or wherein administration is by inhalation or oral administration and the concentration is 0.05 to 10 mg/kg. 
     
     
         23 . A glycoside for use according to any of  claims 20-22 , wherein the concentration of a metabolite of the glycoside is measured up to 300 minutes after administration of the glycoside. 
     
     
         24 . A method of reducing the cell permeability of a glycoside-based exogenous volatile organic compound (EVOC) probe, comprising the step of
 applying conditions that promote conversion of a methyl ester glycoside into a glycoside,   wherein the conditions that promote formation of the glycoside comprise hydrolysis of the methyl ester glycoside in the presence of a base in water.   
     
     
         25 . Use of a glycoside according to  claim 16  or a composition according to any of  claims 17-19  in a breath test for the detection or prognosis of a disease state. 
     
     
         26 . Use according to  claim 25 , wherein the breath test is for the detection of cancer, optionally lung cancer. 
     
     
         27 . A method for the detection or prognosis of cancer comprising assessing the activity of a cancer-specific enzyme by measuring the concentration of an exogenous substrate for said enzyme and/or measuring the concentration of a metabolite of said substrate in a biological matrix of a subject, wherein said exogenous substrate is a glycoside according to  claim 16 . 
     
     
         28 . A method according to  claim 27 , comprising the step of administrating a glycoside according to  claim 16  or a composition according to any of  claims 17-19  to the subject. 
     
     
         29 . A method for determining the activity of an enzyme comprising;
 administering an exogenous substrate for an enzyme to a subject,   measuring the concentration of a metabolite of said substrate in a biological sample that has been obtained from said subject;   wherein said exogenous substrate is a glycoside according to  claim 16 .   
     
     
         30 . A method for monitoring the progression of cancer in a subject diagnosed with cancer comprising:
 administering an exogenous substrate to a subject,   assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject;   wherein said exogenous substrate is a glycoside according to  claim 16 .   
     
     
         31 . A method for determining efficacy of a treatment comprising in a subject diagnosed with cancer:
 administering an exogenous substrate to a subject,   assessing the activity of a cancer-specific enzyme by measuring the concentration of a metabolite of said substrate in a biological sample obtained from the subject wherein said subject has received anti-cancer treatment;   wherein said exogenous substrate is a glycoside according to  claim 16 .   
     
     
         32 . The method according to any one of  claims 27-31 , wherein the glycoside is D5-ethyl-BD-glucuronide. 
     
     
         33 . The method according to  claim 32 , wherein the metabolite is D5 ethanol. 
     
     
         34 . The method according to any one of  claims 27-33 , wherein the cancer-specific enzyme is β-glucuronidase, 
     
     
         35 . The method according to any one of  claims 27-34 , wherein the biological matrix is selected from blood, urine or exhaled breath, for example exhaled breath. 
     
     
         36 . The method according to any one of  claims 27-35 , wherein the glycoside is administered at a concentration of 0.05-10 mg/kg. 
     
     
         37 . The method according to any one of  claims 27-36 , wherein the concentration of the metabolite is measured at up to 300 minutes after administration of the glycoside. 
     
     
         38 . The method according to any one of  claims 27-37 , wherein administration is intravenous administration and the concentration is 0.5 to 10 mg/kg or by inhalation or oral administration and the concentration is 0.05 to 10 mg/kg. 
     
     
         39 . The method according to any one of  claims 27-38 , wherein the concentration is 1 mg/kg to 5 mg/kg, for example about 2 mg/kg. 
     
     
         40 . The method according to any one of  claims 27-39 , wherein the concentration of the metabolite is measured at 10 to 30 minutes after administration of the glycoside. 
     
     
         41 . A glycoside for use according to any of  claims 21-23  or a method according to any of  claims 27-40 , comprising the step of correlating the results with beta-glucoronidase expression, for example as measured by ImmunoHistoChemistry (IHC). 
     
     
         42 . A kit comprising a glycoside according to  claim 16  or a composition according to any of  claims 17-19 , and optionally instructions for use. 
     
     
         43 . A kit according to  claim 42 , further comprising a device for capturing a biological matrix sample from a patient, preferably wherein the device is suitable for capturing a sample of blood, urine or exhaled breath.